Epigallocatechin gallate (EGCG) suppresses growth and tumorigenicity in breast cancer cells by downregulation of miR-25

Abstract

The aim of the present study was to investigate the anticancer effects and potential mechanisms of polyphenol epigallocatechin-3-gallate (EGCG) on breast cancer MCF-7 cells in vitro and in vivo. Our results showed that EGCG significantly inhibited MCF-7 cell viability in a time- and dose-dependent manner. Flow cytometry analysis indicated that EGCG induced apoptosis and disrupted cell cycle progression at G2/M phase. Moreover, EGCG inhibited miR-25 expression and increased PARP, pro-caspase-3 and pro-caspase-9 at protein levels. Restoration of miR-25 inhibited EGCG-induced cell apoptosis. Furthermore, EGCG suppressed tumor growth in vivo by downregulating the expression of miR-25 and proteins associated with apoptosis, which was further confirmed by a reduction of Ki-67 and increase of pro-apoptotic PARP expression as determined by immunohistochemistry staining. These findings indicate that EGCG possesses chemopreventive potential in breast cancer which may serve as a promising anticancer agent for clinical applications.

Keywords: Polyphenol epigallocatechin-3-gallate; apoptosis; breast cancer; proliferation.

Figure 1.

Antiproliferative effect of EGCG on MCF-7 cells (a). MCF-7 cells were treated with 0.5,5 and 20 μg/ml EGCG over a 24–72 h time course. Crystal violet assays were performed as described in the Methods section (* p < 0.01 vs. control at each time point). (b) After 24 h of exposure of MCF-7 cells to EGCG (20 μg/ml), the medium was replaced with fresh serum-free medium without EGCG. The assay was continued for 72 h to compare the effects of EGCG withdrawal with those of continuous EGCG exposure. (*P < 0.05 vs. EGCG withdrawn).

Figure 2.

EGCG induces cell cycle arrest at G2/M phase in MCF-7 cells. (a) MCF-7 cells were treated with EGCG at 0, 5 and 20 μg/ml for 48 h. Cell cycle was analyzed by flow cytometry. (b) Quantified histograms demonstrated the effect of EGCG on MCF-7 cell cycle distribution.*p < 0.05; as compared with control group.

Figure 3.

EGCG induces miR-25-dependent MCF-7 cell apoptosis. MCF-7 cells were treated with EGCG at 5 and 20 μg/ml for 72 h or transfected with mimic miR-25 before treatment with 5 and 20 μg/ml EGCG for 72 h. (a) Cell apoptosis was detected by Annexin V/PI assay; (b) Cellular proliferation was detected by crystal violet DNA staining.

Figure 4.

EGCG inhibits miR-25-dependent MCF-7 cell survival. MCF-7 cells were treated with EGCG at 5 and 20 μg/ml for 72 h. (a) Expression of miR-25 was detected by qRT-PCR assay; (b) Cell apoptosis was detected by Annexin V/PI assay; (c) Cellular proliferation was detected by crystal violet DNA staining. (d) MCF-7 cells were transfected with mimic miR-25 before treatment with 5 and 20 μg/ml EGCG for 72 h. Cellular proliferation was detected by crystal violet DNA staining.*P < 0.05.

Figure 5.

EGCG inhibits tumor growth in MCF-7 xenograft nude mice. (a) Growth curve of tumor volume in nude mice. (b) Expression ofmiR-25 in xenograft tumors as determined by qRT-PCR. (c) Expression of ki-67 in xenograft tumors as determined by IHC. (d, c) Expression of cleaved PARP in xenograft tumors as determined by IHC. (d) TUNEL analysis of xenografts after in vivo EGCG treatment. Data are presented as mean ± SE of three independent experiments, *p < 0.05, as compared with control group.